Bacteria have developed intricate mechanisms to sense changes in their environment and adapt accordingly. The molecular mechanisms that translate an environmental input to defined physiologic outputs are not always well understood, however recent research has uncovered the nucleotide cyclic di-GMP (c-di-GMP) as a global bacterial second messenger that regulates a wide variety of behaviors important to human health such as biofilm formation, pathogenicity, virulence, cell-cell signaling, and cell cycle regulation. Proteins that create, degrade and sense intracellular levels of c-di-GMP are found in the genomes of countless bacteria, including human pathogens. One such conserved c-di-GMP sensing system is the LapD/LapG system. LapD is a transmembrane receptor that regulates the activity of the periplasmic cysteine protease LapG by sensing cytoplasmic c-di-GMP levels through a still poorly understood mechanism. LapG from the model organism Pseudomonas fluorescens has been identified as a protease for a large adhesion protein called LapA that is essential for biofilm formation, suggesting the conserved LapD/G receptor system plays important roles in regulating biofilm formation and adherence to host cells. However, genomic comparisons across important pathogens such as Legionella, Bordetella and Vibrio that possess the LapD/LapG receptor system have failed to identify a conserved substrate of LapG, thus bringing into question the functional output and physiological purpose of this receptor system in these pathogens. The purpose of this proposal, therefore, is to elucidate the molecular mechanism of this receptor system and its functional role in pathogenesis. Specifically, we propose to structurally characterize the full-length transmembrane LapD receptor in the active and autoinhibited states through a variety of techniques including X-ray crystallography, electron paramagnetic resonance, multi-angled light scattering and electron microscopy (Specific Aim 1). The anticipated results will provide unprecedented molecular-level details of a novel inside-out signaling receptor and reveal how conformational changes in the cytoplasm are transduced via LapD's HAMP domain to the periplasm. We will also develop an innovative assay to identify the substrate(s) and substrate specificity of LapG orthologs by introducing photo-crosslinking non-canonical amino acids (Specific Aim 2). These experiments will identify substrates of LapG orthologs and uncover the conserved role of the LapD/LapG system in pathenogenesis. Together, this proposal outlines a unique opportunity to reveal the potential of therapeutically targeting this and other c-di-GMP signaling pathways to ameliorate chronic diseases caused by pathogenic bacteria.